A simple cross‐species method for molecular sexing of softshell turtles ( Trionychidae )

Accurate sex identification is essential for aquaculture, breeding management, and conservation, particularly in species lacking external sexual dimorphism during early development. Softshell turtles (family Trionychidae ) are of high economic importance: their meat is prized in markets, their shells are used in traditional medicine, and large‐scale farming supports rural economies. Male Chinese softshell turtles grow faster than females; therefore, developing reliable sex identification methods for these species is critical. Softshell turtles possess evolutionarily stable ZZ/ZW sex chromosomes, which provide a strong genomic foundation for molecular sexing. In this study, we developed a PCR‐based method for molecular sexing across 5 Trionychidae species, targeting intronic sequence variation in the Ran gene. A single band (~250 bp) is amplified in ZZ males, while two distinct bands (~250 and ~600 bp) are produced in ZW females, due to size differences in the second intron. This diagnostic pattern enables clear and reliable sex identification. Using this method with a conserved primer pair, we successfully sexed individuals of the Chinese softshell turtle ( Pelodiscus sinensis ), Spiny softshell turtle ( Apalone spinifera) , and Florida softshell turtle ( Apalone ferox) . The assay is rapid and cost‐effective and offers a cross‐species approach for molecular sexing across softshell turtle lineages and a practical tool for aquaculture, selective breeding, and conservation genetics.